Microbiological oxidation of pregnenolone to progesterone



United States Patent MICROBIOLOGICAL OXIDATION OF PREG- NENOLONE TOPROGESTERONE David Perlman, Princeton, N.J., assignor to Olin MathiesonChemical Corporation, New York, N.Y., a corporation of Virginia NoDrawing. Continuation of application Serial No. 249,387, October 2,1951. This application November 29, 1957, Serial No. 699,512

6. Claims. (CL, 1951-51) 7 This, application is a continuation of myparent application Serial No. 249,387, filed October 2, 1951, now

abandoned. v This invention relates toa biosynthetic method for thepreparation of progesterone, involving controlled oxidation by means ofthe enzymes of Streptomyces (actinomycetes). 1

More specifically, the process ofthe invention involves themicrobiological oxidation of pregnenolone to progesterone by means ofthese microorganisms, some of which simultaneously produce recoverableantibiotics and/or other medicinals as cobalamines (e.g. vitamin2,915,439 Patented Dec. 1, 1959 during fermentation, for example by theconventional methods of (1) exposing a large surface of the medium toair or (2) utilizing submerged, aerated culture.

Relatively short periods of enzyme action (e.g. fermentation) arerequired for conversion to progesterone,

the optimum time being determined simply from the B )--especially whenthe culture medium is supplemented with assimilable cobalt. Themicrobiological oxidation may be effected by either (1) includingpregnenolone in an aerobic culture of the microorganism, or (2) bringingtogether, in an aqueous medium, pregnenolone, air and enzymes ofnon-proliferating cells of the microorganism.

As the culture medium there may be employed any nutrient medium suitablefor the propagation of the microorganism, such media essentiallycomprising an assimilable source of carbon and energy, and a source ofnitrogenous and growth-promoting factors. The sources of nitrogeneousand growth-promoting factors may be those normally used in fermentationswith these microorganisms. Thus, they may be natural organics (e.g.,soybean meal, corn-steep liquor, meat extract and/or distillerssolubles), or synthetics, such as nitrates, ureas and ammoniumcompounds. The sources of carbon and energy may be (1) fats, (2) fatacids having at least 14 carbon atoms, (3) carbohydrates, or (4)mixtures of the above-mentioned materials. Among the utilizable fats arelard oil, soybean oil, linseed oil, cottonseed, oil, peanut oil, coconutoil, corn oil, castor oil, sesame oil, crude palm oil, fancy muttontallow, sperm oil, olive oil, tristearin, tripalmitin, triolein andtrilaurin. Stearic,

palmitic, oleic, linoleic and myris'tic acids exemplify the utlizablefat acids, while the utilizable carbohydrate sources include suchproducts as starch, sucrose, glucose, maltose, dextrose or impurecompositions containing them. Since it is desired that the pregnenoloneused to be converted to pregesterone rather than utilized by themicroorganism as a source of carbon and energy, the caloric requirementsof the microorganism should be adequately supplied by the other sourcesof carbon and energy mentioned.

Streptomyces generally may be employed as the source of enzymes for theprocess of the invention. Among the utilizable Streptomyces areStreptomyces species ATCC 11,009 (cf. application Ser. No. 239,0l8,filed July 27, 1951, now Patent No. 2,709,705, granted May 31, 1955),Streptomyces aureofaciens, Streptomyces griseus, Streptomyces fradiae,Streptomyces lavendulae, Streptomyces progesterone titer of the mediumand generally being not more than about 48 hours. On extending theperiod of enzyme action beyond this optimum time, the progesteroneformed is converted to further oxidation prodnote. The progesteroneobtained by the process of this invention maybe employed, withoutfurther purification, for the production of 160t-hYdI'OXYPIOg6StCIOIlBby subjecting it to the action (or further action) of the enzymes ofStreptomyces species ATCC 11,009 (cf. Patent No. 2,709,705).

The following specific examples are presented as illustrativebut notlimitativeof the invention:

Example 1 An aqueous medium of the following composition is prepared:

One hunc lred milliliter portions of the medium are distributed in 500ml. Erlenmeyer flasks, and 20 mg. of pregnenolone is added to eachflask. The flasks are then plugged with cotton and sterilized in theusual manner (by autoclaving). When cool, each flask is inoculated with3 ml. of a vegetative inoculum of Streptomyces aureofaciens (NRRL 2209),grown on a soybean mealglucose medium. The flasks are then placed on areciprocating shaker (120 four inch cycles per minute) and agitatedthereon at 25 11 C. for 2 days. The pH of the whole culture is thenadjusted to 3-4 with sulfuric acid, and the solids are removed bycentrifugation.

The aqueous fraction is then extracted 3 times with an equal volume ofchloroform. The chloroform extracts are then pooled, and the solvent isremoved by evaporation under vacuum. An almost quantitative yield ofprogesterone is obtained. The product may be characterized by means ofthe following procedure: An aliquot is chromatographed on filter paperusing a toluenepropylene glycol system [method of Zafiaroni, Science111: 6 (1950)]. After a one-hour development period, the paper stripsare dried and the position of the steroid is spotted using theZimmermann reaction. The steroid present in a duplicate strip is theneluted with ethanol and examined, using a Beckman spectrophotometer, fora marker absorption maximum at 2400 A.; Ecf. method of Samuels et al.,Science 113: 490 (1951)].

The following results are obtained when the procedure PregnenoloneProgesterone added to mefound after indlum, rng./l. cubatlon, mg./l.

Examples 2-6 The procedure of Example 1 is followed except that theStreptomyces aureofaciens (NRRL 2209) is replaced by the following otherStreptomyces, with the results indicated:

Example Progesterone No. Streptomyces yield, percent (approx.)

2 Streptomyces argenteolus 95 3 Streplomyces griseus (Waksman CultureCollection No. 3478M" 90 4 Streptomyces ri-mueus (N RRL 2234 95 5Streptomyces grz'seus (Waksman Culture Collection No. S. griseus 4) 956.; Streptomyces fmdiae (Waksman Culture Collection No. 3535) 85 Example7 The procedure of Example 1 is followed except that the pregnenolone isadded to the growing culture after fermentation has proceeded for oneday. An almost quantitative yield of progesterone is obtained.

The following results are obtained when the procedure of Example 7 isrepeated with larger quantities of pregnenolone.

Preguenolone Progesterone added to mefound after indium, mg./l.cubation, mg./l.

Examples 8 and 9 The procedure of Example 7 is followed except that theStreptomyces awreofaciens is replaced by the following otherStreptomyces, with the results indicated:

Example Progesterone No. Streptomyces yield. percent (approx.)

. Streptomyces argenteolus 95 Streptomyces grz'seus (Waksman CultureCollection N o. S. grz'seus 4) 95 Example 10 Two-day cultures ofStreptomyces aureofaciens (NRRL 2209), grown on a soybean meal-glucosemedium, are centrifuged, re-suspended in distilled water, recentrifugedand re-suspended in distilled water, to yield a suspension containing 23mg. of solids per milliliter. Forty-milliliter aliquots of thesuspension are distributed in 125 Progesterone yield (mm/1.) alterindicated period of Pregnenolone added, mgJliter enzyme action 6 hours24 hours 48 hours The invention may be variously otherwise embodiedwithin the scope of the appended claims.

What is claimed is: I

1. A microbiological process which comprises bringing together, in anaqueous medium, pregnenolone, air and enzymes of a Streptomyces andrecovering progesterone therefrom.

2. A microbiological process which comprises cultivating a Streptomycesunder aerobic conditions in an aqueous nutrient medium containingpregnenolone, a1- lowing the fermentation to continue until at least asubstantial amount of progesterone is formed, and recovering theprogesterone therefrom.

'3. The process of claim 2 wherein the fermentation is allowed tocontinue for a period of not more than about 48 hours.

4. The process of claim 2 in which the medium contains, as at least apart of its source of carbon and energy, a member of the groupconsisting of (1) fat acids containing at least 14 carbon atoms and (2)fats.

5. A microbiological process which comprises bringing together, in anaqueous medium, pregnenolone, air and enzymes of a Streptomyces of thegroup consisting of Streptomyces aureofaciens, Streptomyces argenleolus,Streptomyces griseus, Streptomyces rimosus, Streptomyces fradiae,Streptomyces olivaceous, Streptomyces Iavendulae and Streptomycesvenezuelae and recovering progesterone therefrom.

6. The process of claim 5 in which the medium contains, as at least partof its source of carbon and energy, a member of the group consisting of(1) fat acids containing at least 14 carbon atoms and (2) fats.

References Cited in the file of this patent UNITED STATES PATENTS OTHERREFERENCES Bergey: Manual of Determinative' Bacteriology, 6th ed., p.38.

1. A MICORBIOLOGICAL PROCESS WHICH COMPRISES BRINGING TOGETHER, IN ANAQUEOUS MEDIUM, PREGNENOLNONE, AIR AND ENZYMES OF A STREPTOMYCES ANDRECOVERING PROGESTERONE THEREFROM.